AT414047B - Arrangement for binding molecules, e.g. useful in fluorescence microscopy studies, comprises individual functional groups or multiple identical functional groups arranged on a solid support at a defined density - Google Patents

Abstract

Arrangement for binding molecules comprises individual functional groups or multiple identical functional groups arranged on a solid support at a density of 10 4>-10 1>0>functional groups per cm 2>such that there are no further functional groups within a predetermined radial distance from any given functional group in at least 95% of cases. Independent claims are also included for: (1) producing an arrangement for binding individual molecules by providing a solid surface with functional groups, contacting the surface with auxiliary structures having units (especially organic groups, nucleic acids or polypeptides) capable of binding to the functional groups, treating the surface with agents that either inactivate functional groups not bound to auxiliary structures or block units not bound to functional groups, treating the surface with agents that cleave the linkage between the auxiliary structures and the bound units or fragments thereof, and removing the cleaved auxiliary structures; (2) producing an arrangement for binding individual molecules by providing a solid surface with functional groups and contacting the surface with auxiliary structures having units with activatable functional groups that can bind to the surface functional groups when activated by external stimuli, or providing a solid surface with activatable functional groups and contacting the surface with auxiliary structures having units with functional groups, applying an external stimulus to the surface so that the activated functional groups bind to the other functional groups, treating the surface with agents that cleave the linkage between the auxiliary structures and the bound units or fragments thereof, and removing the cleaved auxiliary structures; (3) producing an arrangement for binding individual molecules by applying nanoscopic islets onto the surface of a solid substrate by a surface structuring method, especially scanning tunneling microscopy, dip pen nanolithography, electron beam lithography, ion beam lithography or microcontact printing, applying adapters to the islets, coupling individual molecules to the adapters and blocking any unoccupied islets with adapter variants not bearing molecules; (4) producing an arrangement for binding individual molecules by applying nanoscopic islets onto the surface of a solid substrate by a surface structuring method, especially scanning tunneling microscopy, dip pen nanolithography, electron beam lithography, ion beam lithography or microcontact printing, coupling molecules to adapters in solution, removing uncoupled adapters or uncoupled molecules by purification steps and applying the molecule-coupled adapters to the substrate.

Description

2

AT 414 047 B

The present invention relates to arrangements for binding molecules, in particular for use as biochips or for analysis by means of single-dye tracing. Method.

The emerging areas of research such as genomics and proteomics pose major challenges for biotechnology, with single-molecule microscopy playing an important role. Common methods of genomics and proteomics are based on investigations using DNA or protein arrays, the evaluation of which determines the ensemble properties of many biomolecules (Arbeitman et al., 2002, MacBeath and Schreiber, 2000, Pollack et al., 1999, Zhu et al., 2001). On the other hand, single-molecule microscopy offers a qualitative advantage of fundamental importance since individual biomolecules can be investigated without their relevant properties being distorted or thinned out by averaging the ensemble observation (Clausen-Schaumann et al., 2000, Mehta et al., 1999; Nie and Zare, 1997; Schmidt et al., 1996; Segers-Nolten et al., 2002; Xie and Lu, 1999). Examples of the properties of single molecules are, for example, the respective set of mutations of a DNA molecule, or the individual post-translational modification of each individual expressed protein, as well as its association with other proteins.

What is needed is a technology that allows for the rapid and unique investigation of many different biomolecules or their structural variants, for example properties of DNA-20 molecules or the type and number of messenger RNA molecules that are expressed in a particular state of the cell or after certain treatment (expression profile); Furthermore, the detailed profiles of the respective expressed proteins, i. Profiles differentiated by type, number, post-translational modification (phosphorylation, glycosylation, etc.), distribution of the individual proteins in the cell, specific protein-protein associations, and the effect on the activity of the protein.

In order to make a statistically relevant statement, the number of single molecules that can be examined at the same time should reach at least a few orders of magnitude, for example 106 to 10®, with a reasonable data acquisition time. 30 For this application, the use of the fluorescence microscopic SDT method is recommended. SDT has been a routine technique for several years, both for detecting individual fixed or diffusing molecules on surfaces (Schmidt et al., 1996) and individual biomolecules in cells (Schutz et al., 2000, Sonnleitner et al., 1999). For ultrafast microscopy of molecules on surfaces with simultaneous single-molecule sensitivity, a method has been developed and patented, " SDT scan " (Hesse et al., 2002, Schindler, 2000). This method allows the analysis of all individual fluorescently labeled molecules to 1 cm2 in about 5 min with a very good signal-to-noise ratio, thus satisfying one of the requirements: providing a sufficiently sensitive and sufficiently fast detection method.

The remaining requirements relate to the development of a methodology for the defined arrangement of the high-density molecules with the maximum value of 1.1 × 10® isolated molecules or an equal number of groups of the same molecules on one cm 2. 45

Currently used methods for arranging molecules on surfaces are used for the production of oligonucleotide, DNA, protein, oligosaccharide and chemical microarrays. Common methods for the preparation of DNA microarrays are based on contact spotting (Schena et al., 1995) and non-contact spotting (Okamoto et al., 2000) low liquid volumes; For oligonucleotide microarrays, photolithographic synthesis is also used on the solid substrate (Fodor et al., 1993). For protein microarrays, spotting methods (MacBeath and Schreiber, 2000) and stamping methods using flexible plastics (Bernard et al., 1998, Bernard et al., 2001) and, additionally, photolithographic synthesis methods are also available for oligopeptide microarrays. Pellois et al., 2002). For Oligosaccha-55 rid microarrays (Fukui et al., 2002) and microarrays of small molecules (Lam and Renil, 2002) 3

AT 414 047 B mostly spotting methods are used.

The spot densities of the microarrays mentioned depend on the production method and range from a few 1000 to 5 × 10 4 spots per cm 2 for the spotting method, and up to a maximum of 5 × 10 5 spots per cm 2 in photolithographic methods.

WO 00/06770 A discloses biomolecule arrays which, although they maintain certain distances between the biomolecule spots, allow only an insufficiently low density of spots for many applications. Furthermore, a specific addressability of the molecules there or the provision of several, different functionalities is not possible. Incidentally, the arrays disclosed there cause problems with respect to nonspecific bonds and the reusability of these arrays is not given.

Although the arrays 15 based on S-layers with proteins as spacers proposed in W089 / 09406 and WO98 / 39688 have a very high density, these arrays are unsuitable for optical analysis for a number of reasons: The distances between the functionalities which are in the range of 10 nm are too small to allow optical resolution; the layers are labile and can not be reproduced. Furthermore, no different functionalities, in particular addressable functionalities, 20 can be provided.

The object of the present invention is therefore to avoid, in whole or in part, the above-described disadvantages of the prior art and to provide arrays of biomolecules which have a high density whose individual binding regions are at a sufficient distance from adjacent binding regions. which can be used in optical analysis systems, can have addressable binding sites or can use different functionalities. In particular, these arrays should be useful for single molecule analysis, especially using the SDT method, and should meet the criteria required for this. 30

Accordingly, the present invention relates to an assembly for binding molecules comprising binding functionalities which are present as molecular-single functionalities or in groups of identical functionalities on a solid support, the density of the individual functionalities or groups of functionalities on the solid support of 35 104 to 1010 individual or grouped bindable functionalities per cm2 is at least 95%, in particular at least 99%, of the individual or grouped bindable functionalities within a selected distance d of any (individual) bondable functionality or group of bondable functionalities no further binding functionalities are located. 40

According to the invention, functionalities are always understood as functionalities that can be linked.

The devices according to the invention represent a decidedly improved alternative to the conventional 45 microarrays, in particular according to the invention densities of 1 × 10 8 molecules or groups of the same molecules or more can be achieved. The minimum distance between molecules or molecule groups, which should still be individually resolvable with fluorescence-optical detection, is approximately equal to the wavelength of the fluorescence light, -0.5 μm (diffraction limit of imaging optical microscopy). According to the invention, this basic limit determines in practice the minimum distance between the molecules or molecule groups of-1 pm, i. there should be no other molecules in an area with a diameter of - 1 pm around each molecule or group of molecules. Nevertheless, the number of molecules or groups should be as high as possible, i. in most cases, at least in the percent range of the preferred maximum areal density of 1 x 10 8 / cm 2 to accentuate the rapid data acquisition of the SDT-scan method. 4

AT 414 047 B

With the present invention, it is thus possible to achieve densities which are at least 100 times greater than e.g. in WO 00/06770 A; Furthermore, the arrangement according to the invention can be provided as an ordered array (in contrast to the "random arrays" according to WO 00/06770 A and WO 98/39688 A). Finally, different specificities of the binding sites can be provided and thus multi-component arrays are provided, which are also still reusable.

According to a preferred embodiment of the present invention, in the arrangement, the distance d is from 0.1 to 100 μm, in particular 0.5 to 10 μm.

These distances allow a particularly efficient use in conjunction with optical methods, in particular with SDT, in particular in the scan mode according to the WOOO / 25113 A.

The arrangement according to the invention preferably additionally has units which are bound to the solid surface via the bondable functionalities, wherein the units are preferably selected from the group consisting of nucleic acids, in particular RNA and DNA, and oligopeptides and polypeptides, in particular antibodies, or organic molecules , in particular members of a combinatorial library.

In the arrangement according to the invention, the density of the individual or grouped bindunas-capable functionalities on the solid support is preferably from 105 to 109, in particular from 10 6 to 10, individual or grouped bondable functionalities per cm 2.

Preferably, the solid surface is a glass, plastic, membrane, metal, or metal oxide surface.

According to a preferred embodiment, the arrangement according to the invention additionally comprises units which are bound to the solid surface via the bondable functionalities and to which molecules, preferably non-covalently, are bound.

According to a further aspect, the present invention relates to a method for producing a device for binding single molecules, which is characterized by the following steps: preparation of a solid surface with bondable functionalities, contacting of the solid surface with auxiliary structures, the units, in particular organic groups , Nucleic acids or polypeptides capable of binding to the bondable functionalities of the solid surface such that the auxiliary structures are attached to the solid surface via the units and functionalities, treating the solid surface with the auxiliary structures attached thereto with agents which either ( i) inactivate the binding functionalities that are not associated with the auxiliary structures so that these bondable functionalities lose their binding capacity, or (ii) the units that do not respond to the bindable functionality - Treating the solid surface with the attached auxiliary structures with means which cleaves the bond between the auxiliary structures and the bonded units or fragments of the bonded units, and - detachment of the auxiliary structures leaving behind the previously of the Auxiliary structures carried units or fragments of these in the region of the contact point on the solid surface.

Preferably, as means for (i) inactivating the binding functionalities and (ii) blocking the units, chemical substances are used which covalently bind to the binding functionalities or moieties and thereby inactivate or block them. Examples are chemical substances with free thiol groups, which can bind to bindable functionalities, such as maleimides. 5

AT 414 047 B

Alternatively, this method of making a device for binding molecules of the present invention may also be accomplished by the following steps: providing a solid surface with functionalities, contacting the solid surface with auxiliary structures carrying units with activatable functionalities that interfere with the functionalities binding the solid surface, if activated by external stimuli, - or providing a solid surface with activatable functionalities and contacting the solid surface with auxiliary structures carrying units with functionalities, - acting on the solid surface with the ones disposed thereon Auxiliary structures, so that the activated functionalities of the auxiliary structures or the solid surface bind to the other functionalities of the solid surface or the auxiliary structures and thus the auxiliary structures via the units to the f bonding the surface, treating the solid surface with the auxiliary structures attached thereto with means which cleave the bond between the auxiliary structures and the bound units or fragments of the bound units, and detaching the auxiliary structures leaving behind the units or fragments previously carried by the auxiliary structures this in the area of the contact point on the solid surface.

Both variants use one and the same solution idea, by means of auxiliary structures a surface is specifically and locally defined activated or deactivated and thus the inventively required parameters of the arrangements can be realized.

Preferably, external stimuli, such as electromagnetic waves in the form of UV light and changes in temperature, are used to activate the activatable functionalities.

Preferably, substantially spherical structures are used as auxiliary structures, which consist of organic or inorganic polymers, metals or metal compounds.

In order to provide a plurality of different specificities and to ensure an addressability of the array, the auxiliary structures preferably have a marking, wherein preferably more than one type of marked auxiliary structures is used.

It is particularly advantageous if auxiliary structures are used with a fluorescence label, and preferably auxiliary structures are used with different fluorescence labeling.

Preferably, any auxiliary structure carries only one specific type of moiety, such as organic groups, nucleic acids, or polypeptides, using a population of substructures each having different occupied units.

According to a preferred embodiment, both the specific fluorescence labeling of the different auxiliary structures and the specific assignment of the auxiliary structures with units before applying the auxiliary structures to the solid surface are known in the method according to the invention.

Preferably, due to the knowledge of the positions of the fluorescence-labeled auxiliary structures on the solid surface, the chemical identity of the units left by the auxiliary structures after the detachment of the auxiliary structures is known.

The contacting of the solid surface with the auxiliary structures preferably takes place with the aid of gravity, centrifugal force, magnetic force, electrical attraction, enrichment at two-phase boundary layers or combinations thereof. 6

AT 414 047 B

Preferably, a glass, plastic, membrane, metal, or metal oxide surface is used as the solid surface.

Particularly preferred bondable functionalities in the context of the present invention are NH 2, SH, OH, COOH, CI, Br, I, isothiocyanate, isocyanate, NHS ester, sulfonyl chloride, aldehyde , Epoxide, carbonate, imidoester, anhydride, maleimide, acryloyl, aziridine, pyridyl disulfide, diazoalkane, carbonyl-diimidazole, Cärbodiimid-, disuccinimidyl carbonate, hydrazine, diazonium, aryl -Azid-, benzophenone, diazirine groups or combinations thereof proven.

Preferably, a spacer molecule can be provided between the bondable functionalities and the units to be bound or between the solid surface and the bondable functionalities.

In a particular embodiment of the method according to the invention, the following steps are used to prepare a device for binding molecules: providing a solid surface with binding functionalities, preferably maleimide functionalities, contacting the solid surface with auxiliary structures, the units, in particular nucleic acids or polypeptides, have linked via molecular recognition and which carry terminal thiol functionalities that can bind to the solid surface such that the auxiliary structures are bound to the solid surface, treating the solid surface with the auxiliary structures bound thereto with thiol-containing reagents, preferably beta-mercaptoethanol which inactivate the functionalities, in particular the maleimide functionalities, which are not associated with the auxiliary structures, - treating the solid surface with the associated auxiliary structures with a physical chemical or chemical stimulus, in particular at elevated temperature, which reverses the molecular recognition between the auxiliary structures and the bonded units, and - detachment of the auxiliary structures, leaving the previously supported by the auxiliary structures units in the region of the contact point on the solid surface.

According to a further particular embodiment of the method according to the invention, the following steps are used to produce a device for binding molecules: providing a solid surface with functionalities, in particular amines, contacting the solid surface with auxiliary structures, the units, in particular organic molecules of a combinatorial library, with activatable functionalities, in particular photoactivatable phenylazides, which can bind to the functionalities, in particular amine functionalities, of the solid surface, insofar as they are activated in particular by a UV light illumination step, the units having chemically cleavable functionalities, in particular disulfide Bridges, are bound to the auxiliary structures, - localized exposure of an external stimulus, in particular UV light, on the solid surface with the auxiliary structures arranged thereon, so that the units in particular bind with the photoactivated phenylazides to the functionalities, in particular the amine functionalities, and thus the auxiliary structures are bound to the solid surface, wherein the external stimulus, in particular in the form of an evanescent field of UV light, is used and localized to functionalities at the solid surface acts as well as on those parts of the auxiliary structures that are in the area of the evanescent field, - treating the solid surface with the auxiliary structures attached thereto with agents, in particular dithiothreitol, which binds the disulfide bridges between the auxiliary structures and the bound units or fragments of the Splitting units, and detaching the auxiliary structures leaving the 7 previously supported by the auxiliary structures

AT 414 047 B

Units or fragments of these in the area of the contact point on the solid surface.

Of course, the invention also relates to arrangements obtainable by the method according to the invention. 5

The arrangements according to the invention are in accordance with another aspect of the invention in the context of a fluorescence microscopy study (especially for single-molecule studies), in particular the "Single Dye Tracing". (SDT) method used. In particular, the arrangements according to the invention can be used for the binding of biomolecules, in particular for the binding of antigens, ligands, proteins, DNA, mRNA, toxins, viruses, bacteria, cells or combinations thereof, and then all possible methods with these arrangements (eg detection and analysis methods). Furthermore, the inventive arrangements for assaying cDNA from cells wherein fluorescently labeled cDNAs bind to the array of different oligonucleotides and the binding can be read for each bound cDNA species are used.

The inventive arrangements are also preferably used to study the proteins 20 of cells, where fluorescently labeled proteins can bind to the array of different antibodies and the binding can be read for each bound protein species.

Realizing a ~ 1 pm diameter free area around each " binding site " (one-half "catcher molecule" or a group of like "catcher molecules") at face densities of " binding sites " in the range of preferably 106 to 108 / cm 2 represents a preferred embodiment of the present invention.

Another feature of preferred arrangements according to the invention is the realization of the addressability of the binding sites. For the intended applications, the position and number of binding sites should be as accurate and a priori known, as well as the type of molecules on each binding site. Such addressability of the binding sites with respect to both position and function (specific molecular recognition of an analyte molecule) is the key to both simple and broad application. For the inventive arrangement with e.g. Antibodies and oligonucleotides are known against which protein which antibody is directed at which site and which oligonucleotide hybridizes with which DNA or messenger RNA sequence at which site of the matrix. Without addressability, no correlation can be established between observations at a binding site and the nature of the bound molecule. No molecule-specific statement would be possible, and thus the central advantage of the application of single-molecule microscopy to the problems described would be omitted. Via addressability, samples of many different biomolecules can be broken down by labeling with only one fluorescence marker, since fluorescence detection at a specific site means binding to a known capture molecule. Without functional addressability, the analyte molecules would need to be either individually labeled and sequentially measured, or many different dyes used for color-specific labeling simultaneously, either impracticable or reaching practical limits very quickly. 50

The addressability not only facilitates access to information, but also the computing time for data acquisition and evaluations is postponed within a reasonable range. In fact, without prior knowledge alone, the positions of the binding sites would take a long time to calculate the computation time with fast computers and fast algorithms, 55 since the necessary search of the molecular positions (maxima of single-molecule fluorescence intensity)

AT 414 047 B) of ~ 108 molecules is computationally intensive.

The requirements underlying the invention for the realization of a molecular matrix (hereinafter abbreviated to "MARS, Matrix of Addressable Recognition Sites") can be summarized as follows:

The binding sites for molecular recognition (capture molecules, either singly or in groups of the same) should be identified on the " MARS " preferably in the following way in combinations: 1. Isolated detectable (no molecules in the radius of min. ~ 0.5 μm binding site) 2. Position of each binding site should be known a priori 3. Function of each binding site should be known a priori 4. Die Density at different addressable binding sites should be high (10® to 108 / cm2).

Accordingly, the invention relates in particular to the fabrication of templates of organic molecules or biomolecules (eg, antibodies, receptors, peptides, oligonucleotides, or nucleic acids) which are transferred and bound via suitable ancillary structures to a substrate surface such that the biomolecules transferred are isolated or isolated Isolated groups of the same molecules are present, ie isolated observable with optical single-molecule microscopy. Both the position and the nature of each individual biomolecule or of each isolated group of identical molecules is known in advance, whereby the mean surface density of the bound molecules should reach very high values. The application of these addressable molecular matrices serves to quantify the specific binding (molecular recognition) of binding partners (eg antigens, ligands, proteins, DNA, messenger RNA, toxins, viruses, bacteria, cells, etc.), using the fluorescence Labeling of the binding partners and a new, patented microscopy method (Single Dye Tracing, SDT), which allows the reading of these molecule templates with high sensitivity (imaging down to single fluorescence molecules) and very high speed.

In the following, preferred implementations of the arrangements according to the invention will now be described with reference to the embodiments sketched schematically in the drawing figures.

Auxiliary structures (FIG. 1A), in particular spheres (3, 11) of organic or inorganic materials with diameters (18), which depend on the intended use and in the range of ~ 0.5 pm to ~ 100, may be used to fulfill the objects set forth herein pm lie. Each sphere (3,11) carries " catcher molecules " (4, 6) and only one of each type. By adding the spheres to a substrate (1), the " catcher molecules " (5, 7, 12, 13) in contact with the substrate surface and a controlled transfer (8, 14) of " catcher molecules " (5, 7, 12, 13) from the spheres (3, 11) on the substrate surface, such that binding sites (individual or groups of like "catcher molecules") are formed, which are spatially separated from one another (9, 10, 15, 16). FIG. 1B outlines the individual steps of the transmission process by way of example on a transmitted " catcher molecule ". The " catcher molecules " (eg, antibodies, oligonucleotides, etc., symbol Y in FIGURE 1B) are bound to the beads, preferably via molecular recognition by complementary biomolecules (eg, epitopes for antibodies, complementary oligonucleotides, etc., symbol X in FIGURE 1B) are bound to the spherical surface directly or via flexible spacer molecules. The spheres are preferably added in the liquid phase to the substrate and are deposited on the surface thereof. Under suitable conditions, a hexagonal packing can be achieved. Their nonspecific interaction with the substrate is weak compared to the specific binding of the " catcher molecules ". The " catcher molecules " (4, 6) bind (Figure 1B: 5, 7, 12, 13) covalently or via specific noncovalent molecular interaction to the surface of the substrate (1). The bond strength of a " catcher molecule " is enough to hold the sphere at the binding site. After fixation of " catcher molecules " to the substrate, the balls are removed from the surface (36) and the " catcher molecules " remain at the point of contact with the ball on the substrate (9, 10, 15, 16). Upon detachment of the spheres, the molecular recognition complexes between the transferred " catcher molecules " and dissociate the complex molecules bound to the spheres. The molecular recognition bindings can be easily selected by selecting specific liquid phase conditions without harming the " catcher molecules " be solved without the fixation of " catcher molecules " is undone by the substrate. Other methods of detachment are the use of tensile forces (using magnetic balls (Edelstein et al., 2000)) or hydrodynamic shear forces. 10

The " prints " left by the bullets are either single " catcher molecules " (FIG 1A: 9, 10) using spheres of correspondingly low population density (FIG. 3), or groups of M capture molecules (FIG. 1A: 15, 16), using spheres of correspondingly high density bonded " capture molecules " (11). In both cases, the minimum distance 15 of the binding sites (2) given by the diameter of the balls (18), reduced by the diameter of the binding region (17). The achievable density of binding sites corresponds to the density of the transfer spheres, which can reach a hexagonal dense packing. However, the addition of spheres and their random attachment via " catcher molecules " not reaching the maximum density, however, about 25% of the maximum density is easily achievable, a density high enough for all applications. For example, with d = 1 pm spheres, approximately 30 million binding sites are transferred at this density per cm2 (intended normal area of the molecular matrix).

The number of per &quot; impression &quot; transferred &quot; catcher molecules &quot; depends on several factors 25 and can be controlled by it. Two important factors are the areal density of &quot; catcher molecules &quot; on the sphere as well as the binding capacity of the substrate to the &quot; catcher molecules &quot;. In addition, the number of transmitted &quot; catcher molecules &quot; also influenced by the size of the contact surface (FIG. 1A: 17) between ball (11) and substrate (1). 30 A larger contact area results when using substrates that are coated with a compressible polymer layer. When using a coating using linear flexible polymer chains, the radius of the contact region (17), r, can be calculated from the sphere diameter (18), d, and the effective polymer length, h, with the relationship r 2 ~ h * d. For the flexible spacer molecule PEG (polyethylene glycol), Mw = 2000 with a maximum elongated length 35 of 15 nm, measured values for its flexibility (Jeppesen et al., 2001) result in a dynamic length range of h = 5 -10 nm for a sphere with a diameter (18) of d = 1 pm, a bond region (17) with the radius r = 70-100 nm. High occupation density spheres (11), M &quot; catcher molecules &quot; pro r2, then allow the transfer of about M &quot; catcher molecules &quot;. M can be adjusted in a wide range using established methods for the occupation of spheres with biomolecules via molecular recognition, with densities of up to 1/50 nm2. For example, with a sphere of d = 1 pm, a group of M &quot; catcher molecules &quot; adjustable, adjustable between M = 1 and M - 200, with a statistical deviation of M1 / 2. 45 For transmission (FIG. 1A: 8) of individual &quot; catcher molecules &quot; (9, 10) are bullets with low population of &quot; catcher molecules &quot; (3) used. The reliability of single-molecule transfer can easily be estimated for experimentally controllable conditions. The mean number of &quot; catcher molecules &quot; on the ball, let <N>. At low values of &lt; N &gt; random distribution can be assumed, both the number N of &quot; catcher molecules &quot; on individual spheres (Poisson distribution with mean <N>) as well as the distribution of N &quot; catcher molecules &quot; on the surface of each sphere, the probability of finding M of the N &quot; catcher molecules &quot; is described in the contact area by the binomial distribution. This provides, without further assumptions, a formula for estimating the error probability that more than one &quot; catcher molecule &quot; P (&gt; 1) = 55 (&lt; N &gt; * (r / d) 2) 2. For &lt; N &gt; = 3, a ball diameter (18) d = 1 pm, and a diameter 10

AT 414 047 B (17) of the effective contact area for 2r = 200 nm binding (conservative value, see above) is P (> 1) ^ 0.0009, i. at most every 1000th ball transmits two "catcher molecules". The fact that, statistically, no &quot; catcher molecule &quot; has no significant influence on the result (slight reduction in the mean density of the binding sites). For even less occupation of the balls, for example for &lt; N &gt; = 1, the reliability of transmission of individual &quot; catcher molecules &quot; (at most every 10000th binding site carries two &quot; catcher molecules &quot;), but also the proportion of spheres without &quot; catcher molecules &quot; rises to -37%. io To check the position of the imprints of the transmitted &quot; catcher molecules &quot; At the substrate, the positions of the spheres are measured prior to their detachment (Figure 2: 22). Again, the use of the SDT-scan method is advantageous. When using fluorescent spheres, the position of each sphere from its image (24) on the pixel array (23) of the CCD (Charged Coupled Device) camera used in the SDT method can be determined very accurately, 15 to about 40 nm (Hesse et al., 2002). For practical applications, the more inaccurate assignment of individual pixels to each sphere (FIGS. 2: 26 and FIG. 3: 26) is sufficient, as a result of which the limiting time of the data evaluation is substantially shortened to about the time of the data acquisition. In addition to the spheres used to make binding sites, the matrix also contains spheres that serve as position references (Figures 2: 19 and 3: 19). These spheres 20 carry reactive functionalities (FIG.2: 20) in high density and are fixed to the substrate through multiple covalent or noncovalent bonds (FIG.2: 21) and can not be peeled off. They are fluorescent and give a significantly larger signal (FIG.2: 25) on the pixel array (FIG.2: 23). Upon detachment of the transfer spheres, the reference spheres remain on the substrate and their positions (FIGURE 2: REF 1) give a long-lived 25 frame to retrieve (FIGURE 3:27) the positions of the binding sites (FIGURE 3: 26) ). The SDT-scan method allows you to retrieve every position of a surface that is only 0.1% covered with randomly distributed reference spheres, with a precision well below a pixel, even after interim removal of the matrix from the SDT-scan microscope. In order to realize the functional addressability (a priori assignment of type and number of "catcher molecules" to the binding sites) color coding of the spheres is used (FIG. 4). Color coding of balls of inorganic or organic polymers of the size in question is state of the art and is offered in some variants by companies (Bangs, Luminex, Microparticles, micromod, dynal). Here are fluorescent molecules 35 of different colors in the spheres, whereby the numbers of the molecules of each color can be set to defined graded values. With n different colors and m different concentrations of each fluorophore, in principle, mn-1 distinguishable in their fluorescence spheres can be produced. By binding certain &quot; catcher molecules &quot; on spheres of particular color code, fluorescence measurement of the bound spheres for each bin site (Figure 4: 26) measures a color code (33, 34) indicating which &quot; catcher molecule &quot; present at the binding site. Due to the extreme sensitivity of the SDT method, spheres can be accurately measured for their levels of fluorescence intensity and position. In FIG. 4, this is outlined for 5 grades 0, 1, 2, 3, and 4 (commercially available up to 10 grades for the same dye) of fluorescence from 4 different dyes. For each color, the fluorescence image of the sphere matrix (FIG. 4) is recorded separately. This yields for each sphere position (Figure 4: 26) a color code (33), for example 1314 for the sphere at location 2, 7, determined from the 4 different fluorescence intensities (29, 30, 31, 32) for the latter Bullet. With the 4 colors and the 5 gradations 54 -1 = 624 different balls can be distinguished. The optical resolution of the SDT-scan method presumably allows the distinction of libraries with thousands of different color-code spheres, but the creation of such large libraries requires considerable technical effort. For each sphere of the matrix, the pixel position and the color code including those of the high fluorescence intensity reference spheres (34) are stored. This data set, after detachment (Figure 5: 36) of the transfer 55 beads, serves to identify the binding sites within the matrix. The ~ 1 cm2 matrix 1 1

AT 414 047 B (FIG. 5: 37) of binding sites and reference spheres with a diameter of 1 μιτι together with the stored positions Pi of all binding sites, i = 1 to * -30 million, and their function (type of &quot; catcher molecule &quot; Fi, and number of &quot; catcher molecules &quot;, mi, at the point i), is said to be &quot; MARS (Pi; Fi, mi) &quot; where &quot; MARS &quot; the abbreviation of &quot; Matrix of Addresses 5 Sable Recognition Sites &quot; is.

The use of spheres to transfer molecules, in addition to producing a biochemical "Matrix of Addressable Recognition Sites", MARS, also permits the production of the chemical analogue, a "Matrix of Addressable Chemical Reaction Sites", MACS for short. In contrast to MARS, in the production of MACS, the detachment of the beads is not accomplished by dissociation of complementary biochemical binding partners such as oligonucleotides, but by the chemical cleavage of a covalent bond. FIG. Figure 6A shows the steps involved in the preparation of a MACS exemplified by a molecule. The molecules to be transferred are bound to the beads and are composed of a cleavable functionality (40) (e.g., a disulfide bridge), an optional moiety (57, 58), and a terminal functionality. After addition, the beads attach to the substrate and bind to the surface via the terminal functionalities. To detach the beads, the cleavable functionalities (40) are screened (e.g., reduction of the disulfide by dithiothreitol); a portion of the cleaved functionality (41, 42) (e.g., thiol) as well as the optional moiety 20 (60, 61) remain on the substrate (1). MACS Molecule matrices can be prepared by the route described herein for two different purposes: In the first case (FIG.6B-1), it is intended to have a population of different molecules (57, 58) of spheres (3, 11) on the planar substrate to transfer (59). The different molecules are present either individually (60) or in groups (61) in the matrix MACS (Pi; Fi, mi), where Pi is the position, Fi the molecule type and mi the number of molecules for the binding sites, i = 1 to 30 million, is. An example of a specific application is a combinatorial library of organic substances on spheres (Jung, 2000, Nicolaou et al., 2002). One sphere carries only one type of organic substance at a time and all of the various substances are to be transferred to a planar substrate in order to test their biological activity.

In order not to disturb the evaluation, the cleaved functionalities (41, 42 in FIG.6A and FIG.6B-1, e.g., thiol) may be inactivated (methyl iodide). 35

In the other case (Figure 6B-2), a chemical matrix having identical, cleaved reactive functionalities (41, 42) (e.g., thiol) is prepared on a surface. The chemical functionalities can be further chemically modified, such as with antibodies or oligonucleotides. In general, this chemical matrix can be described by MACS (Pi; mi) where 40 is the number mi of reactive functionalities at position Pi. FIG. 7 sums up some ways, such as &quot; Matrices of Addressable Chemical reaction Sites &quot; of the last-mentioned type MACS (Pi, mi) (FIG. 6B-2) and then set in MARS &quot; Matrices of Addressable Recognition Sites &quot; can be transferred. Starting from the substrate (1), the functionalities (40) of spheres (11, 3) with high or low occupation density are transferred to the substrate surface via the transfer step (44, 46). The resulting templates contain isolated sites with groups of reactive functionalities (41), MACS (Pi, mi) (45), or single reactive functionalities (42), MACS (Pi, 1) (48). Chemical matrix MACS (Pi, mi) (45) can also be transferred to MACS (Pi, 1) (48) (47) by applying smaller fertilized spheres (38) with very few reactive functionalities (40). This offers the advantage of being able to produce high-purity matrices with individual reactive functionalities (42). This is achieved by cyclic repetition of binding of the smaller spheres (38) with functionalities (40) (eg, ortho-pyridylsulfide) to some of the reactive functionalities (41) (eg, thiol), which are thereby protected from subsequent inactivation of the free functionality -55 onalities on the substrate (1) eg by N-ethyl maleimide. In this way (47) it does not come to 1 2

AT 414 047 B shows a substantial reduction in the surface density of the reactive sites (42). Of course, the same procedure can also be applied directly to the matrix &quot; MACS (Pi, 1) &quot; (48) (not included in FIG. 7) to cleanse the matrix of defects with more than one reactive functionality. 5

The resulting chemical matrices (45, 48) are universally replaceable and can be used to make &quot; MARS &quot; matrices of various kinds. Some examples are shown. Way (51): the preparation of multi-functional single-molecule matrices, &quot; MARS (Pi; Fi, 1) &quot; (55), is facilitated by the fact that the loading of the balls, advantageously small balls io (39) with &quot; catcher molecules &quot; (4, 6) in high occupancy, the area density of the &quot; catcher molecules &quot; on the balls, because for their binding to the substrate surface, only one reactive group on the substrate is available per binding site. Way (50): Alternative way to make &quot; MARS (Pi; Fi, 1) &quot; (55), using smaller, with fewer &quot; catcher molecules &quot; (4, 6) loaded balls (38). Path (52): matrices with only one type of &quot; catcher-15 molecule &quot;, &quot; MARS (Pi; 1, 1) &quot; (56) can be directly obtained by adding &quot; catcher molecules &quot; be generated in the liquid phase (53). Path (49) is one way of making matrices &quot; MARS (Pi; Fi, mi) &quot; (54) with single or groups of capture molecules. Path (49) starts from the matrix MACS (Pi, mi) (45) and is an alternative to the direct way (43) to make &quot; MARS (Pi; Fi, mi) &quot; Matrices without chemical matrices. 20

The invention will be explained in more detail with reference to the following examples and the drawings, to which it is of course not limited.

Show: 25

Fig. 1: (A) Schematic representation of the process in which individual or groups of molecules are transferred by means of spherical auxiliary structures to a planar substrate. (B) Schematic representation of the individual steps of the process of transfer of biomolecules exemplified by a "catcher molecule". 30

Fig. 2: Schematic representation of attached to a planar substrate balls whose fluorescence intensity is determined by scanning.

Fig. 3. Schematic representation of the method to uniquely determine the position of fluorescent spheres 35 using an array of pixels.

Fig. 4. Schematic representation of the fluorescence images of surface-localized and color-coded beads containing different fluorophores in different concentrations. 40

Figure 5. Schematic representation for making a &quot; Matrix of Addressable Recognition Sites &quot;, MARS.

Figure 6. Schematic representation of the process of making a "Matrix of Addressable Chemical Reaction Sites", MACS using Spherical Auxiliary Structures. (A) Representation of the individual steps of the process for producing a MACS by way of example by means of a molecule. (B) Schematic representation of the production of two different types of MACS.

Figure 7. Schematic representation of various ways to &quot; Matrices of Addressable Chemical &quot; as reaction sites in &quot; Matrices of Addressable Recognition Sites &quot; convert.

Fig. 8: Applications for MARS matrices: Utilization of high sensitivity.

The large extent of the template "MARS (Pi; Fi, 1) &quot; of ~108 binding sites allows many different "catcher molecules". (here exemplarily 100 different antibodies, it can also 1 3

AT 414 047 B

Mixtures of oligonucleotides and antibodies are used) and at the same time each &quot; catcher molecule &quot; in many copies (here 1 million), resulting in the detection sensitivity of individual fluorescence molecules, a dynamic range for detection of 6 orders of magnitude, simultaneously for the detection of 100 different 5 biomolecules, measurable with SDT scan in ~ 5 min ,

Fig. 9: Applications for MARS matrices: proteomics.

Fig. 9A: Applications for MARS templates: protein function. Simultaneous measurement of the radio-tion of many (here 104) individual proteins (triangles) by repeated image acquisition, to capture the currently bound ligand (L), which carry a fluorescent label (star). The ligands in solution give a negligible background fluorescence in the preferred application. Application to enzymes is particularly promising. The functional responses (series of +, binding, and -, not binding) 15 directly yield the binding constants and the association and dissociation constants for ligand binding of each of the 104 receptor molecules or enzymes, thereby allowing the direct measurement of functional variability of biomolecules. The SDT-scan method allows to record 104 molecules at ~ 1 pm intervals in 50 ms, so that the proteins function with a time resolution of -50 ms. 20

Fig. 9B: Applications for MARS matrices: measurement of post-translational modification of proteins. By using fluorescently labeled antibodies against phosphorylation sites (P) and labeled lectins against sugar residues on the proteins, the profile of these modifications for one or more proteins can be measured, and optionally with the previously measured variation of function (Figure 9A) into one Structure-function relationship can be put.

Figures 9C & D: Applications for MARS Matrices: Measurement of Protein Associations. It is exploited that the SDT method allows stoichiometric measurements of co-localized fluorescence molecules. The intensity at the binding sites provides information on homoassociation (Figure 9C) or heteroassociation (Figure 9D). For the latter case, it is shown in the lower part of the picture that the same information can be achieved or checked and secured independently by the use of two different dyes. 35 Fig. 10: Applications for MARS matrices: DNS and mRNA analyzes.

Fig. 10A: Applications for MARS matrices: measurement of mRNA profiles. A matrix of numerous different oligonucleotides, but each in a sufficiently high number to ensure high sensitivity, can be used to generate mRNA profiles of, for example, 40 extracts of cells or organelles for different states of the cells.

Fig. 10B: Applications for MARS matrices: Correlation of adjacent SNPs. There is currently a great deal of interest in determining the order of single nucleotide poly-morphism (SNP) in certain regions of the genome. The DNA single strands in question are first fixed to magnetic spheres with one end and the other end to corresponding oligonucleotides on the matrix. By applying magnetic force, the DNA strands are stretched so that the binding of fluorescently labeled oligonucleotides to the SNP sites to be examined is as safe as possible. The fluorescence response by SDT scan then provides information on mutations occurring at the SNP sites. 50

Fig. 10C: Applications for MARS matrices: mutations in &quot; repeats &quot;. The same procedure as in Figure 10B can be applied to the identification of mutations in regions of DNA with repeats (repeats) using two different oligonucleotides: one against the natural sequence and one against the mutated sequence , The upper half of the picture shows the result for DNA without mutation, the lower 55 5 5 1 4

AT 414 047 B with a mutation.

11: FIG. 1: Visualization of individual fluorophores by means of the SDT-scan method.

Fig. 12: Figure to Example 2: Hexagonal dense packing of balls on a surface.

FIG. 13: FIG. 3: Replacement of marker beads. A: Arranged Mar kerbeads before (A) and after detachment (B) from uncoupled beads. 10

FIG. 14: FIG. 4 shows the impression of groups of fluorescently labeled molecules which have been transferred by means of spheres to a planar surface and bound covalently.

FIG. 15: FIG. 5 shows beads arranged on a surface with different fluorescence markings.

Index of the designations in FIGS. 1-7: I. Substrate 20 2. Minimum distance of the transferred molecules from the substrate 3. Sphere for transfer, with few capture molecules occupied 4. Scavenger molecule of defined type for transfer, bound to a sphere by molecular recognition 5. An 6. The capture molecule of defined type for transfer, bound on a sphere by molecular recognition, capture molecule different from 4 7. Capture molecule (6) fixed to the substrate before detachment of the sphere 8. PROCEDURE Transferring individual catcher molecules (4) after detachment of the sphere 30 10. Transferred individual capture molecules (6) after detachment of the sphere II. Sphere for transfer, with many capture molecules occupied. 12. Capture molecules fixed to the substrate (groups of 4) before detachment of the sphere 13. capture molecules fixed to the substrate (groups of 6) Detachment of the sphere 14. PROCEDURE: Transfer of several capture molecules (groups) 35 15. Transferred groups of capture molecules (4) after detachment of the sphere 16. Transferred groups of capture molecules (6) after detachment of the sphere 17. Bonding area at the contact surface sphere Substrate 18. Diameter of a sphere for transfer 19. Reference sphere 40 20. High density reactive groups on reference sphere 21. To the substrate &quot; covalently bonded &quot; PROCEDURE: Scanning the Balls Prior to Detachment 23. Pixel Array 24. Image of Balls for Transfer to Pixel Array 45 25. Image of Reference Balls on Pixel Array 26. Position of the binding sites defined by position of the spheres imaged on the pixel array 27. PROCEDURE: Position recovery 28. Fluorescence intensity of the reference sphere 29. Fluorescence intensity of a defined sphere for transmission, within a selected wavelength range. 30. Fluorescence intensity of a defined sphere for transmission, within a 29 wavelength range. 31. Fluorescence intensity of a defined sphere for transmission, within a wavelength range deviating from 29 and 30. 55 32. Fluorescence intensity of a defined sphere for transmission, within one of 29, 30 1 5

AT 414 047 B and 31 deviating wavelength range. 33. Color code (with arbitrary values) 34. Color code of the reference sphere 35. Content remaining after spheres after cleavage of the functionality 40 5 36. PROCEDURE: detachment of the spheres for transfer 37. Matrix of binding sites and reference spheres, MARS (Matrix of Addressable Recognition Sites) or MARS (Pi, Fi, mi) 38. Small spheres for transmission, with few molecules occupied 39. Small spheres for transmission, with many capture molecules occupied io 40. Reactive cleavable functionalities for the generation of MACS (Matrix of Addressable Chemical reaction Sites)

41. Transmitted groups of reactive functionalities on MACS

42. Transmitted single reactive functionalities on MACS 43. PROCESS for the generation of MARS (Pi, Fi, mi) (37) 15 44. PROCESS for the generation of MACS (Pi, mi) (45) 45. MACS with groups of equal functionalities, MACS (Pi, mi) 46. PROCESS for the generation of MACS (Pi, 1) (48) 47. PROCEDURE for the transfer of MACS (Pi, mi) (45) to MACS (Pi, 1) (48) 48. MACS with individual PROVIDING MACS (Pi, mi) (45) in MARS (Pi, Fi, mi) (54) 50. PROCEDURE FOR TRANSFERRING MACS (Pi, mi) (45) in MARS (Pi, Fi, 1) (55) 51. PROCEDURE for the transfer of MACS (Pi, 1) (48) to MACS (Pi, Fi, 1) (55) 52. PROCEDURE for the transfer of MACS ( Pi, 1) (48) in MACS (Pi, 1,1) (56) 53. Liquid phase capture molecules 25 54. MARS (Pi, Fi, mi) with individual and groups of different capture molecules of different types, from MACS ( Pi, mi) (45) produced 55. MARS (Pi, Fi, 1) with only individual catcher molecules of different types Kind, made of MACS (Pi, mi) (45) and MACS (Pi, 1) (48)

56. MARS (Pi, 1,1) with only single capture molecules of the same species, made from MACS 30 (Pi, 1) (48) 57. Molecule of defined type for transmission, bound on a sphere 58. Molecule of defined type for transmission, on tied to a ball; Molecule different from 57 59. PROCEDURE: Transfer of molecules individually and / or into groups 35 60. Transferred single molecule 61. Transferred group of molecules

Examples: 40 Example 1:

Single Cy3 fluorophores were immobilized on a glass slide and visualized by SDT scan. For immobilization, Cy3 was first coupled to polyethylene glycol diamine, and this conjugate was covalently attached to a 45 epoxide surface via the remaining terminal amine group of the PEG. To prepare the Cy3-PEG conjugate, 0.2 g (100 pmol) of PEG-diamine (Mw 2000, Rapp Polymerere) was dissolved in 4 ml of hydrochloric acid dimethylformamide (DMF) pH = 6.5 and treated with 1 mg (1.3 pmol ) Cy3-N-hydroxysuccinimide (Cy3-NHS, Amersham Pharmacia) dissolved in 1 ml of DMF, incubated for 3 hours. The reaction was started by adding 100 μl of a 5% disopropylethylamine (DIEA) / DMF solution; Reaction course and reaction end were monitored by thin layer chromatography (TLC). Purification was via gel filtration and ion exchange chromatography, and the purity of the fractions was checked by TLC. To produce glass slides with epoxy coating, coverslips (Esco, microscope cover glass, 24 x 50 mm, Erie Scientific, Portsmouth, NH) were etched with trifluoroacetic acid and glycidoxypropyltrimethoxysilane 55 (GPS) according to the publication (Hehler et al., 2000). silanized. The quality of the coating 16

AT 414 047 B was confirmed with contact angle measurements (Dataphysics OCA 20). The coupling of Cy3-PEG-amine to the silanized glass slides was carried out at a concentration of 20 nM (measurement with UV-Vis spectrophotometer Shimadzu UV1601) in 50 mM borate buffer pH = 8.8 for 10 minutes. Excess unbound Cy3-PEG amine was removed by washing several times in water. To visualize the immobilized fluorophores, the fluorescence reader &quot; nanoreader &quot; used. For excitation, a diode-pumped solid-state laser with a wavelength of 532 nm was used, as an objective Axiovert PNF 40x / 1.3 oil was used. The exposure time was 100 ms and the signal was filtered with a Cy3 filter set (Chroma, HQ610_75m). For picture reproduction the program io V ++ (Digital Optics) was used. Figure 11 shows an area of 30 pm by 50 pm with single and cluster of fluorophores with an average single signal intensity of 15 counts. The signal to noise ratio was 50 on average.

Example 2: 15

Beads with a coating of polyethylene glycol (PEG) were used to produce a hexagonal packing of beads on a surface. For coating with PEG, the beads (latex beads, Polysciences, diameter 2 pm, carboxyl surface) were suspended in 20 μl of 1M 2- (N-morpholino) ethanesulfonic acid (MES) buffer to a final concentration of 20 1% by mass and mixed with 1 ml of a solution of 0.1 M MES buffer pH = 4.5 with 26 mM 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and 10 mM methoxy-polyethylene glycol-amine (Mw 5000, Shearwater Polymers, Huntsville, AL, USA) and incubated for 2 hours in a shaker at 150 rpm. After PEG coating, the beads were washed several times and applied to a pegylated glass slide surface 25 in a 1% suspension. To prepare a pegylated glass surface, glycidoxypropyltri-methoxysilane-modified glass slides (preparation see Example 1) with 100 mg of methoxy-polyethylene glycol-amine (Mw 5000) (Shearwater Polymers) in 25 ml of 50 mM borate buffer pH = 8.8 at room temperature for 24 Incubated for hours. Figure 12 shows a 110 pm by 60 pm excerpt of a transmitted light image of hexagonal close packing 30 pegylated latex beads on pegylated glass slides taken with the nanoreader.

Example 3:

To demonstrate the use of marker beads, a mixture of non-fluorescent beads and covalently coupling fluorescent marker beads was applied to a glass substrate. The markerbeads covalently bound and remained on the surface after washing while uncoupled beads were detached from the surface. For the preparation of coupling marker beads, fluorescence-labeled silicate beads (sicastar® -redF from Micromod, with carboxyl surface, diameter of 2 μm) were modified via EDC activation with PEG-diamine (Mw 3400, 40 Shearwater Polymers). For modification, the beads were resuspended in 20 μl of 1M MES buffer pH = 4.5 at a final concentration of 1% by mass. This suspension was transferred to 1 ml of 0.1 M MES buffer pH = 4.5 with 26 mM 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC; Sigma) and 10 mM polyethylene glycol diamine (Mw 3400, Shearwater Polymers ) and incubated for 2 hours in a shaker at 750 rpm 45. The multiple wash was done with water. For the preparation of non-coupling beads, unlabelled silicate beads (sicastar® from Micromod, with carboxylate surface, diameter of 2 μm) in 1 ml of 0.1 M MES buffer pH = 4.5 with 26 mM EDC and 10 mM methoxy. Polyethylene glycol amine (Mw 5000, Shearwater Polymers) for 2 hours in a shaker (Thermomixer comfort, Eppendorf) at 750 rpm incubated. The multiple wash was done with so water. For the occupation experiments with beads, pegylated glass plates with an amine-reactive surface were used. To produce a pegylated glass surface, glycidoxypropyltrimethoxysilane-modified glass slides (preparation see Example 1) were incubated with 100 mg of methoxy-polyethylene glycol-amine (Mw 5000, Shearwater Polymers) in 25 ml of borate buffer pH = 8.8 at room temperature for 24 hours. After washing several times, the terminal amine function was modified with succinic anhydride (Sigma) in a saturated solution maintained at 55 1 7 AT 414 047 β pH = 6.2-6.5 with 6N NaOH for 2 hours. The resulting carboxylate function was activated in 25 ml of DMF with 13 mM N.N.N'.N'-tetramethyl-O-iN-succinimidyl-J-uronium tetrafluoroborate (TSTU) and 6.9 mM NHS with the addition of 20 μM triethylamine. The washing was carried out with a DMF / isopropanol gradient, finally it was dried in nitrogen-5 Ström. To cover the coated glass slides with beads, a mixture of 0.01% marker beads with PEG-amine terminus and 1% beads with PEG terminus in borate buffer pH = 8.8 was added, the mixture was applied to the plate and 60 minutes incubated. Fig. 13 A shows a transmitted light image of the attached beads. During the incubation, the marker beads coupled with the amino-terminus to the NHS-activated glass surface so that the marker beads remained on the glass slide after a wash step while peeling off non-coupling beads. FIG. 13B shows a Cy3 fluorescence image of the adhering Makerbeads after the detachment of the non-coupling beads. The platelet area shown is the same as that in FIG. 13 A. 15 Example 4

Fluorescently labeled molecules were transferred from spheres to a flat surface in a controlled manner. For this purpose, beads were coated with amine-PEG-Cy3 and applied to a glass plate with PEG-NHS coating. At the contact points between beads and glass, 20 amine-PEG-Cy3 was transferred to the glass substrate and covalently coupled, leaving fluorescently labeled imprints after washing the beads. For this transfer, 20 μΙ silicate beads (Bangs Laboratories Inc, with carboxylate surface, diameter 5 μιη) were resuspended in PBS buffer pH = 7.3 with a final concentration of 10 percent by mass. This bead suspension was added to 1 ml of PBS buffer pH = 7.3 with 1.2 .mu.l (1 mg) of amine-PEG-Cy.sub.3 (Syn-25-thete see Example 1) and shaken for 2 hours (Thermomixer Eppendorf comfort). incubated at 750 rpm. After repeated washing in PBS buffer pH = 7.3, no fluorescence signal (fluorescence spectrometer Hitachi f-4500) was detected in the supernatant. Markerbeads (Silicate Beads, Bangs Laboratories Inc., carboxylate surface, diameter 5 pm with a mixed PEG-amine / Cy3-30 PEG-amine surface, preparation analogous to Example 3) were mixed in 1000-fold excess to the loading transfer beads. The mixture was applied to the glass slide with PEG-NHS coating and incubated for 15 minutes (preparation of the coated platelets see Example 3). The transfer beads were peeled off by washing and the fluorescently labeled imprints were visualized with the nanoreader (see Example 1). FIG. 14 A shows an approximately 100 μm by 35 50 μm section with a marker fluorescence intensity of high intensity and approximately 100 steps of lesser intensity. Fig. 14B shows a partial detail of Fig. 14A with impressions of different fluorescence intensities.

Example 5: 40

To demonstrate the use of color-labeled beads, three types of different fluorescence-labeled silicate beads (sicastar® blueF and sicastar® greenF, sicastar® redF from Micromod, with carboxylate surface, diameter 2 pm each) were used. The beads were modified with methoxy-PEG-amine by EDC activation (see Example 2 for preparation). 45 Likewise, the glass substrate was coated with methoxy-PEG-amine (preparation see Example 2). To cover the glass plate with balls, a mixture of the three types of beads with equal proportions of 0.5 mass percent was suspended in water with a total final concentration of 1.5 mass percent and applied to the plate. For fluorescence recording of the adsorbed beads, a fluorescence microscope with a mercury vapor lamp (Zeiss, fluo arc HBO 100) and a lens (Zeiss, Axiovert PNF 40x / 1.3 oil) was used. A picture was taken with three filter sets: DAPI excitation filter: D365 / 10x, dichroic beam splitter: 380DCLP, emission filter: D460 / 50m. FITC excitation filter: HQ480 / 40x, dichroic beam splitter: Q505LP emission filter: HQ535 / 50m. Cy3 excitation filter: HQ535 / 50x, dichroic beam splitter: Q565LP emission filter: HQ610 / 75m. Cy5 stimulation filter: HQ620 / 60x, dichroic beam splitter: Q660LP emission filter: HQ700 / 75m. The

Claims (22)

For the sake of clarity, individual images were color-contrasted depending on the emitted wavelengths and superimposed with the aid of the software V ++ (FIG. 15). The blue balls correspond to the signals of Sicastar blue, the green balls to those of 5 Sicastar red, the black balls to those of Sicastar green. References: Arbeitman et al. (2002). Science 297, 2270-2275. Bernard et al. (1998). Langmuir 14, 2225-2229. Bernard et al. (2001). Nat Biotechnol 19, 866-869. Clausen-Schaumann et al. (2000). Curr Opin Chem Biol 4, 524-530. Edelstein et al. (2000). Biosens Bioelectron 14, 805-813. Fodor et al. (1993). Nature 364, 555-556. 15 Fukui et al. (2002). Nat Biotechnol 20, 1011-1017. Hesse et al. (2002). J Chromatogr B 782, 127-135. Jeppesen et al. (2001). Science 293, 465-468. Jung (2000). Combinatorial Chemistry: Synthesis, Analysis, Screening, Vch Publishing Company). 20 Lam et al. (2002). Curr Opin Chem Biol 6, 353-358. MacBeath et al. (2000). Science 289, 1760-1763. Mehta et al. (1999). Science 283, 1689-1695. Nicolaou et al. (2002). Handbook of Combinatorial Chemistry: Drugs, Catalysts, Materials, John Wiley &amp; Sons). 25 Never et al. (1997). Annu Rev Biophys Biomol Struct 26, 567-596. Okamoto et al. (2000). Nat Biotechnol 18, 438-441. Pellois et al. (2002). Nat Biotechnol 20, 922-926. Piehler et al. (2000). Biosens Bioelectro 15, 473-481. Pollack et al. (1999). Nat Genet 23, 41-46. 30 Schena et al. (1995). Science 270, 467-470. Schindler (2000) WOOO / 25113 Schmidt et al. (1996). Proc Natl Acad Be U S A 93, 2926-2929. Protection et al. (2000). Embo J 19, 892-901. Segers-Nolten et al. (2002). Nucleic Acids Res 30, 4720-4727. Sonnleitner et al. (1999). Bi-35 Ophys J 77, 2638-2642. Xie et al. (1999). J Biol Chem 274, 156967-15970. Zhu et al. (2001). Science 293, 2101-2105. 40 Claims: 1. An arrangement for binding molecules comprising bindable functionalities which are present as molecular-single functionalities or in groups of identical functionalities on a solid support, the density of the individual functionalities or groups of functionalities on the solid support from 101 2 to 1010 individual or grouped functionalities per cm 2, is at least 95%, in particular at least 99%, of the individual or grouped functionalities within a selected radius d of any (individual) bindable functionality or group of functionalities no further binding functionalities are located. 50 2. Arrangement according to claim 1, characterized in that the distance d of 0.1 to 100 pm, in particular 0.5 to 10 pm. An assembly according to claim 1 or 2, characterized in that they additionally comprise units which are bound to the solid surface via the bondable functionalities, the units preferably being selected from the group consisting of nucleic acids , in particular RNA and DNA, as well as oligopeptides and polypeptides, in particular antibodies, or organic molecules, in particular members of a combinatorial library. 5 4. Arrangement according to one of claims 1 to 3, characterized in that the density of the individual or grouped bindable functionalities on the solid support of 105 to 101 2, in particular from 106 to 10®, single or grouped bindable functionalities per cm2. 10 5. Arrangement according to one of claims 1 to 4, characterized in that the solid surface is a glass, plastic, membrane, metal, or metal oxide surface. 6. Arrangement according to one of claims 1 to 5, characterized in that they additionally lent units which are bound to the solid surface via the bondable functionalities, and to which molecules, preferably non-covalently bound. 7. A method for producing a device for binding of single molecules, characterized by the following steps: 20 - providing a solid surface having bondable functionalities, - contacting the solid surface with auxiliary structures bearing units, in particular organic groups, nucleic acids or polypeptides, which can bind to the bondable functionalities of the solid surface, so that the auxiliary structures on the units and Functionalities are bound to the solid surface, 25 - treating the solid surface with the auxiliary structures attached thereto with agents which either (i) inactivate the bondable functionalities that are not associated with the auxiliary structures such that these bondable functionalities lose their binding ability, or (ii) the units which are not bound to the bondable functionalities of the solid surface block, 30 - treating the solid surface with the auxiliary structures attached thereto with means which cleave the bond between the auxiliary structures and the bound units or fragments of the bound units, and - detaching the auxiliary structures leaving behind the units or fragments previously supported by the auxiliary structures in the region the contact point on the solid surface. A method of making a device for binding molecules, characterized by the steps of: providing a solid surface having bondable functionalities and contacting the solid surface with auxiliary structures carrying units with activatable bondable functionalities that conform to the functionalities of the solid surface, provided they are activated by external stimuli; or providing a solid surface with activatable bondable functionalities and contacting the solid surface with auxiliary structures carrying units with functionalities capable of binding; acting on an external stimulus on the solid surface with the auxiliary structures arranged thereon, so that the activated bondable functionalities of the auxiliary structures or the solid surface to the other bondable functionalities of the solid surface or the Hilfstruktu - binding the auxiliary structures to the solid surface via the units, - treating the solid surface with the auxiliary structures bound thereto with agents which cleave the bond between the auxiliary structures and the bound units or fragments of the bound units, and 2 - detaching of the auxiliary structures, leaving behind the units or fragments previously obtained from the auxiliary structures, or in the region of the contact point on the solid surface. 9. The method according to claims 7 to 8, characterized in that as auxiliary structures substantially spherical structures are used which consist of organic or inorganic polymers, metals or metal compounds. 10. The method according to claim 7 to 8, characterized in that the auxiliary structures have a marking and preferably more than one type of marked auxiliary structures are used. 10 11. The method according to any one of claims 7 to 10, characterized in that auxiliary structures are used with a fluorescence label, and preferably auxiliary structures are used with different fluorescence labeling. 12. The method according to any one of claims 7 to 11, characterized in that a beliebi ge auxiliary structure carries only a specific type of units, such as organic groups, nucleic acids or polypeptides, and a population of auxiliary structures is used, each with different occupied units. 13. The method according to any one of claims 7 to 12, characterized in that both the specific fluorescence labeling of the different auxiliary structures and the specific assignment of the auxiliary structures with units before applying the auxiliary structures on the solid surface is known. 14. The method according to any one of claims 7 to 13, characterized in that due to the knowledge of the positions of the fluorescence-marked auxiliary structures on the solid surface, the chemical identity of the units left by the auxiliary structures after the replacement of the auxiliary structures is known. 15. The method according to any one of claims 7 to 14, characterized in that the Kontak animals of the solid surface with the auxiliary structures with the aid of gravity, centrifugal force, magnetic force, electrical attraction, enrichment of two-phase boundary layers or combinations thereof takes place. 16. The method according to any one of claims 7 to 15, characterized in that a glass, plastic, membrane, metal, or metal oxide surface is used as a solid surface. 17. The method according to any one of claims 7 to 16, characterized in that as bindable functionalities NH2-, SH-, OH-, COOH-, CI-, Br-, I-, isothiocyanate-, isocyanate-, NHS ester, sulfonyl chloride, aldheyde, epoxide, carbonate, imidoester, anhydride, maleimide, acryloyl, aziridine, pyridyl disulfide, diazoalkane, carbonyl diimidazole, carbodiimide , Disuccinimidyl carbonate, hydrazine, diazonium, aryl-azide, benzophenone, diazine-irine groups or combinations thereof. 45 18. The method according to any one of claims 7 to 17, characterized in that between the bindable functionalities and the units to be bound or between the solid surface and the bondable functionalities, a spacer molecule is provided. 50 19. The method according to any one of claims 7 and 9 to 18, characterized in that the following steps are used to prepare an assembly for binding of molecules: - Providing a solid surface with bondable functionalities, in particular 55 maleimide functionalities, 5 10 15 20 Contacting the solid surface with ancillary structures that have attached units, in particular nucleic acids or polypeptides, via molecular recognition and which carry terminal thiol functionalities that can bind to the solid surface, so that the auxiliary structures bonding to the solid surface; treating the solid surface with the auxiliary structures attached thereto with thiol-containing reagents, in particular beta-mercaptoethanol, which inactivate the functionalities, in particular the maleimide functionalities which are not associated with the auxiliary structures the firm Oberf with the auxiliary structures attached thereto with a physical or chemical stimulus, such as elevated temperature, which reverses the molecular recognition between the auxiliary structures and the bound units, and detachment of the auxiliary structures leaving behind the units previously supported by the auxiliary structures in the area of the contact point the solid surface. 20. The method according to any one of claims 8 to 18, characterized in that for the preparation of a device for binding of molecules, the following steps are used: - providing a solid surface with functionalities, in particular amines, - contacting the solid surface with auxiliary structures, the units , in particular organic molecules, a combinatorial library, with activatable functionalities, in particular photoactivatable phenylazides, carry, which can bind to the functionalities, in particular to the amine functionalities, the solid surface, if activated, in particular by a UV light illumination step the units are bound to the auxiliary structures via chemically cleavable functionalities, in particular disulfide bridges, - localized action of an external stimulus, in particular UV light, on the solid surface with the auxiliary structures arranged thereon, so that the units in particular with the photoactivated phenylazides to the functionalities, in particular the amine functionalities bind and thus the auxiliary structures are bound to the solid surface, wherein the external stimulus, in particular in the form of an evanescent-the field of UV light, is used and localized Functionalities on the solid surface as well as on those parts of the auxiliary structures that are in the area of the evanescent field - treating the solid surface with the attached auxiliary structures with agents, in particular dithiothreitol, which blocks the disulfide bonds between the auxiliary structures and the attached units or cleaving fragments of the bonded units, and - detaching the auxiliary structures, leaving the units or fragments previously carried by the auxiliary structures or fragments thereof in the region of the contact point on the solid surface. 21. Arrangement according to one of claims 1 to 6, obtainable by a method according to one of claims 7 to 20. 22. Use of an arrangement according to one of claims 1 to 6 in the fluorescence micro-45 roskopieuntersuchung. 23. Use of an arrangement according to one of claims 1 to 6 for the binding of biomolecules, in particular for the binding of antigens, ligands, proteins, DNA, mRNA, toxins, viruses, bacteria, cells or combinations thereof. 24. Use of an assembly according to any one of claims 1 to 6 for the study of cDNA of cells, wherein fluorescently labeled cDNAs bind to the array of different oligonucleotides, and the binding can be read for each bound cDNA species. 55 22 AT 414 047 B 25. Use of an arrangement according to one of claims 1 to 6 for the examination of the proteins of cells, wherein fluorescently labeled proteins bind to the array of different antibodies and the binding can be read for each protein species bound. For this purpose 20 sheets of drawings 10 15 20 25 30 35 40 45 50 55